Purified Anthocyanin , its Elicitation from Cell Cultures of Begonia malabarica and Begonia rex-cultorum ‘ Baby Rainbow ’ and its In vitro Cytotoxicity Analysis by MTT Assay

Background: According to recent statistics, cancer accounts about marked percentage of total deaths in the world, although there are many therapeutic approaches. Unfortunately, the cytotoxicity properties of most chemotherapy drug are nonspecific and therefore do not distinguish between normal healthy cells and tumor cells, these events have led to inappropriate and toxic therapeutic agents with a wide range of side effects. However, several experimental and epidemiological studies have suggested that fruits and vegetables are associated with low risk of various types of cancer. Anthocyanins are natural pigments that provide intense purple to red color in plants. Anthocyanin possess the ability to inhibit oxidative stress and to induce apoptosis in malignant cells, thus may prevent carcinogenesis. Methods: Antiproliferative properties of purified anthocyanin extract from elicited cell suspension cultures of Begonia malabarica and Begonia rex-cultorum ‘Baby rainbow’ was investigated in terms of MTT assay. Anthocyanin extracts were tested for their ability to inhibit the growth of HT29 (colon cancer cells), MG63 (Osteosarcoma), HeLa (Cervical cancer cells) and L929 (Mouse Fibroblast L929) cell lines. Results: Cell viability decreased in a dose dependent manner in all the considered cell lines treated with anthocyanin extracts. The extract of Begonia rex-cultorum ‘Baby rainbow’ exhibited significant cytotoxic activity against all tumor cell lines than Begonia malabarica extract. Begonia malabarica and Begonia rex-cultorum ‘Baby rainbow’ anthocyanin extract exhibited the highest cytotoxicity towards HT29 and HeLa cell lines respectively. But, MG63 resulted in comparatively higher percentage of viability of cell lines at the same concentrations. The anthocyanin extract produced significant morphological alterations on cell lines in culture. Meanwhile, the extracts showed poor cytotoxicity against the normal cell line. Conclusion: The morphological alteration of the treated cancer cells presented clear evidence of significant cytotoxicity of anthocyanin extracts of both Begonias in all the three cell lines. Thus, anthocyanin may act as chemopreventive agents for various cancer cell lines.

roles of anthocyanin as medicinal agents have been well-accepted dogma in folk therapy throughout the world.Secondary metabolites with bioactivity were isolated and used either directly or after chemical modification.Their pharmacological values are increasing due to the constant discoveries of their potential role in healthcare and as lead molecule for new drug designing.Begonia, a perennial flowering plant belongs to the family Begoniaceae, are rich source of anthocyanin content.Synthesis of anthocyanins often requires elicitors, which act as molecular signals in plant stress responses.Elicitation of cell cultures (using chemical or environmental) is a recognized and efficient means of maximizing anthocyanin pigment towards commercialization of product recovery.In both in vitro and in vivo research trials have demonstrated that the marked ability of anthocyanin to reduce malignant cell proliferation and to inhibit tumor formation. 3harmacognosy Journal, Vol 10, Issue 3, May-Jun, 2018 Phytochemicals capable of retarding the cell cycle and/or activating the cellular apoptotic response in the cancerous cells is an attractive trend in antimetastatic studies.Thus, in the present study, a comparison of preliminary antiproliferative potential of the purified anthocyanin from the cell suspension cultures of Begonia malabarica and Begonia rexcultorum 'Baby rainbow' were carried out on selected cell lines.

Plant materials
The fresh healthy plants of Begonia malabarica and B. rex-cultorum 'Baby rainbow' were collected from the wild habitats.Identity was confirmed by referring floras and authenticated with herbaria of Jawaharlal Nehru Tropical Botanical Garden and Research Institute, Palode, Kerala.The voucher specimen was deposited in the herbarium of University College, Trivandrum (UCB 1207, UCB 1208)

Establishment of cell suspension culture
Fresh callus of Begonias species was cultured in a 250 mL flask with 40 mL of modified liquid MS culture medium at (25 ± 1)°C on a rotary shaker with a speed of 100 rotations per min (rpm) under 16 h illumination with 80 μmol m -2 s -1 .The medium was autoclaved at 121°C for 20 min.The cell suspension derived from the 1 g calli tissue from leaf explants of B. malabarica were subcultured in liquid MS culture medium containing 2,4-D (0.1 mg L -1 ), NAA (0.5 mg L -1 ), and BAP (0.5 mg L -1 ) every week until the cells showed continuous and stable accumulation of biomass.Meanwhile, in B. rex-cultorum 'Baby rainbow' (leaf explant) cell suspension was established in liquid MS medium supplemented with the growth regulators such as BAP (1 mg L -1 ) + 2,4-D (0.5 mg L -1 ), BAP (2.0 mg L -1 ) + IAA (1.0 mg L -1 ), KN (2 mg L -1 ) + IAA (1 mg L -1 ) and KN (2 mg L -1 ) + 2,4-D (1 mg L -1 ).After cell culture for one cycle, the cells were harvested by filtration via a Buchner funnel, washed with distilled water to remove residual medium, and filtered again.Then, the weighed fresh cells (FW) were dried at 50°C to constant dry weight (DW).Cell growth was measured based on the FW and DW.Every 20 days, cells were subcultured to fresh media [with 5.0 g fresh weight (FW)] in 250-mL flasks.

Elicitation of anthocyanin in cell suspension culture
Different concentrations of peptone water, yeast extracts, phenylalanine, abscisic acid (ABA), salicylic acid (SA), zinc sulphate and methyl jasmonate (MeJA) were used to study the elicitation effect on anthocyanin production on the cell suspension cultures of B.malabarica (BM) and B. rex-cultorum 'Baby rainbow' (BR).MeJA and SA were dissolved in ethanol and all others were dissolved in water and were filtered before adding into the suspension cultures.Based on the results of preliminary experiments, the concentration employed were peptone water (0.015%), yeast extracts (1%), phenylalanine (10 μM), abscisic acid (0.25mg/L), salicylic acid (60 μM), zinc sulphate (50 μM)) and methyl jasmonate (1 μM).All experiments were performed in triplicate.

Quantification and purification of anthocyanin
Anthocyanin was isolated and quantified from the in vitro cell suspension and in vivo plants.The anthocyanin elicited cell suspension culture and fresh leaves were homogenized in 3 ml methanol with 1% HCl and the extract was used for quantification of anthocyanin by the protocol of Sutharut and Sudarat. 4 The absorbance was read at 510 and 700 nm against distilled water as blank.The combined aqueous concentrates of anthocyanin after evaporation were purified by partition method against ethyl acetate and further purified by Amberlite XAD-7 column chromatography.Then, the adsorbed anthocyanins were eluted using ethanol containing 7% acetic acid (v/v) as mobile phase.
The column eluted fraction with highest anthocyanin content was used for the LC-MS analysis.The experiment was performed on a Thermo Scientific Dionex UltiMate® 3000 RSLC system with chromatographic separation achieved on a Thermo Scientific Acclaim® RSLC 120 C18 reversed-phase column (2.1 × 100 mm, 2.2 µm) operated at 40°C with gradient elution at 0.5 mL/min.The mobile phase consisted of three components: A) acetonitrile, B) deionized (DI) water, and C) 20% formic acid.Mobile phase C was held constant at 10% to provide 2% total formic acid in the mobile phase throughout the run.Mobile phase A was ramped from 0% to 8% from 11 to 42 min, then held for 13 min before returning to the initial composition from 55 to 60 min.Electrospray ionization (ESI) was used as the interface and was operated in positive selected ion monitoring (SIM) mode.The probe temperature was set at 500°C and needle voltage was set at 2000 V.The cone voltage was set at 50 V for all SIM scans with a span of 0.3 amu for each SIM.
In vitro antiproliferative effect determination by MTT assay HT29 (colon cancer cells), MG63 (Osteosarcoma), HeLa (Cervical cancer cells) and L929 (Mouse Fibroblast L929) cell lines were cultured in 25 cm 2 DMEM supplemented with 10% FBS, L-glutamine, sodium bicarbonate and antibiotic solution containing: Penicillin (100 U ml -1 ), Streptomycin (100 µg ml -1 ), and Amphoteracin B (2.5 µg ml -1 ).Cultured cell lines were kept at 37ºC in a humidified 5% CO 2 incubator.The viability of cells was evaluated by direct observation of cells by Inverted phase contrast microscope and followed by MTT assay method.Two days old confluent monolayers of cells were trypsinized and the cells were suspended in 10% growth medium.100µl cell suspension (5x10 4 cells/ well) was seeded in 96 well tissue culture plates and incubated at 37ºC in a humidified 5% CO2 incubator.After 24 h the growth medium was removed, freshly prepared anthocyanin extracts in 5% DMEM were five times serially diluted by two-fold dilution (100, 50, 25, 12.5, 6.25 µg in 1000 µl of 5% MEM) and each concentration of 100 µl were added in triplicates to the respective wells and incubated at 37ºC in a humidified 5% CO 2 incubator.Detectable changes in the morphology of the cells, such as rounding or shrinking of cells, granulation and vacuolization in the cytoplasm of the cells were considered as indicators of cytotoxicity.15 mg of MTT was reconstituted in 3 ml PBS until completely dissolved and sterilized by filter sterilization.MTT assay was carried as per the protocol of Laura et al. 5 The absorbance was measured by using microplate reader at 570 nm.The percentage of growth inhibition was calculated using the formula:

% of viability Mean OD Samples
Mean OD of control group  100

Statistical analysis
Analysis of variance (ANOVA) was used to determine significant differences between treatments, using the statistical package STATISTICA.All the values were mean of 12 replicates ± SE.Significance level was noticed 5%.

Cell suspension culture of B. malabarica and B. rex-cultorum 'Baby rainbow'
Cell suspension culture was established by culturing fresh friable calli from the leaf explants of B. malabarica in liquid MS media supplemented with definite combinations and doses of BAP, 2, 4-D and NAA with concentrations 0.5, 0.1, 0.5 mg L -1 respectively.MS liquid medium supplemented with 2, 4-D (0.1 mg L -1 ) and BAP (0.5 mg L -1 ) combinations showed well established suspension cultures i.e., suspensions without any aggregation or clumps of cells (Figure 1a).The in vitro suspension of cells from B. malabarica revealed optimal and steady biomass accumulation on day 14.The same medium and growth hormone combinations were used for further growth kinetics studies.After 16 th day, cells in the suspension exhibited a reduction in fresh weight as well as dry weight of cells (Table 1).The maximum fresh weight (8.0 g) and dry weight (0.83 g) was noticed at 14 th day of culture in liquid MS medium complimented with 2, 4-D (0.1 mg L -1 ) + BAP (0.5 mg L -1 ).The time course of biomass accumulation was the typical sigmoid growth curves.

Quantification of anthocyanin
Anthocyanin was quantified from the in vitro cell suspension culture and in vivo plants.The in vitro cells showed remarkable level of anthocyanin ie., 10.4 mg and 20.6 mg / 100 ml for Begonia malabarica and B. rex-cultorum 'Baby rainbow' respectively.However, in the in vivo plants anthocyanin content noticed was 5.7mg g -1 for Begonia malabarica and for B. rex-cultorum 'Baby rainbow' 9.8 mg g -1 .
From the given results it can be speculated that anthocyanin content may be effectively induced through in vitro culture by changing the culture parameters.The present results seem to be more effective and supported by other in vitro cultures of phytochemicals from medicinal plants.
Elicitation by chemicals on anthocyanin production B. malabarica and B. rex-cultorum 'Baby rainbow' cell suspension cultures showed poor accumulation of anthocyanin under elicitation using peptone water and yeast extracts.Similarly, the addition of phenylalanine (10 μM), the precursor of secondary metabolite synthesis at lower doses enhanced the anthocyanin synthesis in the cells.Purification and identification of anthocyanin 25 g of fresh, homogenized cell suspension sample was extracted out from the cultures of Begonia malabarica and B. rex-cultorum 'Baby rainbow' .The combined aqueous concentrates after evaporation were purified by partition method against ethyl acetate to remove chlorophylls, stilbenoids, less polar flavonoids and other non-polar compounds from the mixture.The non-aromatic compounds were removed with the use of Amberlite XAD-7 column chromatography.Amberlite XAD-7 adsorbs the aromatic compounds including anthocyanins and other flavonoids in aqueous solutions, whereas free sugars and other polar non-aromatic compounds were removed by washing with distilled water until the eluted water has a neutral pH.Therefore, aqueous acidified ethanol with the concentration of 75% was used for the desorption of anthocyanins from the Amberlite XAD-7HP column.Subsequently, purified amberlite column eluted fraction was used for the antimetastatic analysis.LC-MS/MS was successful in identifying the major anthocyanin fractions of B. malabarica and B. rex-cultorum 'Baby rainbow' .The major anthocyanin fractions from the two species were eluted between 4.

Cytotoxicity analysis by MTT assay
Many vegetables and fruits in the human diet have been proved as potential neutraceutical agents and consuming optimal amounts can inhibit the development of life style diseases.Cancer is one of the major issues Pharmacognosy Journal, Vol 10, Issue 3, May-Jun, 2018     worldwide.Therapeutic herbals have been utilized massively by the local populations as remedial for many disorders and hence, it is need of the hour to evaluate the efficacy in terms of toxicity in using them.Several studies have reported that herbal extracts can be used in the management of oxidative stress.

In vitro cytotoxicity activity analysis against selected cancer cell lines
In the present study, the cytotoxic effect of purified anthocyanin was analyzed using MTT assay on three cancer cell lines (HT29, MG63 and HeLa cell lines) and one normal cell line (Mouse Fibroblast L929).The concentration employed ranged from 6.25 to 50 μg ml -1 .IC 50 level of treatment was prolonged to 48 and 72 h, the % of viability was found to decrease concentration and duration dependently.At the concentrations of 6.25, 12.5, 25 and 50 µg mL -1 of B. malabarica, the % of viability further decreased to 61.5, 53.6, 41.7, 28.2 % respectively in HeLa cells after 72h.
A similar tendency was noticed with HT29 and MG63 cell lines.The results were found to be statistically significant.The concentration and duration dependency of anthocyanin extract may be due to the presence of different anthocyanin fractions that function synergistically together.B. rex-cultorum 'Baby rainbow' also showed a similar trend against HeLa, HT29 and MG63 cell lines (Table 3 a and b)

Cytotoxic effect on Mouse Fibroblast (L929) Cells
No significant cytotoxicity was noticed with the anthocyanin of B. malabarica and B. rex-cultorum 'Baby rainbow' by MTT assay on mouse fibroblast (L929) cells.The 50 values were found to be 14.8 and 5.5 g L -1 respectively.Thus, revealing the selectivity mode of action of anthocyanin i.e. toxic towards malignant cells and safe against fibroblast cells.

Morphological studies
In this part of the study, morphological anomaly if any with anthocyanin treatments was analyzed.No visible morphological changes were noticed for L929 cells with anthocyanin treatments.In fact, HT29, MG63 and HeLa cell lines (Figure 3), showed an increased number of rounded cells and growth inhibition when compared with the untreated control cells.This data further substantiates the MTT results.activity of ethanol extracts from 36 species was evaluated against K562 human leukemia cells.Rhodea japonica and Hypericum chinense were cytotoxic at a concentration of 10 mg ml -1 .The main cytotoxic constituent of Rhodea japonica was isolated and identified as rhodexin A. Nalbantsoy et al. 10 analyzed antimicrobial and cytotoxic activities of Zingiber officinalis extracts.The results of the morphological observation and MTT assay revealed that the cytotoxic activity was dose dependent.IC 50 values versus L929 and HeLa cells were found to be 87.28 µg ml -1 and 74.32 µg ml -1 , respectively, for the chloroform extract, while the ethanol extract showed IC 50 values at 101 µg ml -1 and 33.78 µg ml -1 , respectively.Weisburg et al. 11 compared the in vitro cytotoxicity of epigallocatechin gallate and tea extracts against cancerous and normal cells from the human oral cavity.Negahdari et al. 12 compared wound healing activity of extracts and herbal formulations of Aloe vera, henna, Adiantum capillusveneris on mouse dermal fibroblast cells.All the results substantiate that phytochemicals from plants showed selective toxicity against cells in a varied manner as revealed by the anthocyanin of the present study.

CONCLUSION
In the present study, significant antiproliferative activity was found, which varied among the Begonia species, but the values were acceptable comparing to the international standards.Further work is required to isolate and characterize the individual bioactive fraction of anthocyanin and to evaluate its potentiality.

Figure 2 a
Figure 2 a: LC-MS analysis of B. malabarica.

Figure 3 :
Figure 3: MTT assay of purified anthocyanin extract of Begonia malabarica and B. rex-cultorum 'Baby rainbow' on different cell lines (100 µg ml-1 concentration) along with their control.

Table 1 :
Fresh (FW) as well as dry weight (DW) of cells obtained in suspension culture for first 20 days of B.malabarica in liquid MS medium with 2, 4-D (0.1 mg L -1 ) + BAP (0.5 mg L -1
9hahneh et al.8reported varied in vitro cytotoxic activity by MTT assay of four herbals used in persian traditional medicine on six cancer cell lines such as non-Hodgkin's B-cell lymphoma, human leukemic monocyte lymphoma, human acute myelocytic leukemia, human breast carcinoma, human Prostate Cancer and mouse fibrosarcoma cell lines and one normal cell line; Human Umbilical Vein Endothelial Cells.Masuda et al.9screened medicinal and edible plants in Okinawa, Japan.Cytotoxic anthocyanin was further evaluated at different time intervals i.e., 24, 48 and 72 h against each cell lines.The in vitro cytotoxic activities of purified anthocyanin from Begonia malabarica and B. rex-cultorum 'Baby rainbow' were shown in the Table3a and b and IC 50 values were determined from the dose response curves (Table3a and b).Comparison of purified anthocyanins from the two-species revealed that anthocyanin from B. rex-cultorum 'Baby rainbow' exhibited significant cytotoxic activity against all tumor cell lines with remarkable IC 50 values.However, anthocyanin of B. malabarica showed tumor selective cytotoxic activity depending on the cell line type i.e., HeLa cervical cell were the most sensitive cell line and MG63 was the most resistant tumor cell line (in terms of IC 50 values).Meanwhile, B. rex-cultorum 'Baby rainbow' anthocyanin extract was more cytotoxic against HT29 cell lines followed by MG63 bone cells (Table3a and b).The values obtained were statistically significant.The cytotoxic impact of the anthocyanin was evaluated via MTT assay revealed the cell viability based on the reduction of yellow tetrazolium MTT to a purple formazan dye by the mitochondrial dehydrogenase enzyme.The quantity of formazan produced reflects indirectly the number of metabolically active viable cells.MTT results showed that anthocyanin of both the species displayed cytotoxic effect against cancer cell lines in a concentration dependent manner.Such anti-proliferative potential of anthocyanin was featured by its concentration and tumorselective manner, as reflected by the comparatively low IC 50 values.The IC 50 concentrations of anthocyanin from B. malabarica and B. rex-cultorum 'Baby rainbow' were evaluated against different cell line for 24 h.The IC 50 values of B. malabarica were 35.9, 26.2, 53.2 µg mL -1 against HeLa, HT 29, MG63 cell lines respectively.Meanwhile, B. rex-cultorum 'Baby rainbow' displayed 38.7, 20.4,26.83 µg mL -1 against HeLa, HT 29, MG63 cell lines respectively.Anthocyanin extracts of B. malabarica and B. rex-cultorum 'Baby rainbow' have shown significant inhibition of HT29 cell proliferation as compared to other cell lines.Further, a duration (24, 48, 72 h) and concentration dependent analysis was carried.When the duration of